Publications

Endothelial injury caused by viruses usually involves viral replication or transformation. We report a novel mechanism of endothelial damage by a toxic viral protein. We have isolated a new retrovirus from hemangiosarcomas which appeared among layer hens. The isolated avian hemangiosarcoma virus (AHV) is capable of inducing hemangiomas in hens in-vivo and causes a cytopathic effect (CPE) and loss of thromboresistance in cultured bovine aortic endothelial cells (BAEC). These effects do not require viral replication and can be induced by purified AHV envelope glycoprotein (gp85). AHV causes CPE in BAEC through a typical programmed cell death (apoptosis). Quiescent G0/G1-BAEC are much more sensitive to AHV induced apoptosis than actively dividing cells. These experiments demonstrate the capacity of viral proteins to affect the integrity and functionality of vascular endothelial cells.

Hemangiomas are localized tumors of vascular cells which appear frequently in humans and animals, and their mode of induction is unknown. Recently, a new field strain of avian retrovirus, avian hemangioma virus (AHV), was isolated from spontaneous hemangiomas in layer hens. Sequence analysis of the AHV genome revealed the presence of three prototypic retroviral genes, gag, pol, and env, but no oncogenes. AHV was capable of inducing hemangiomas in hens in vivo, but it induced a strong cytopathic effect in cultured endothelial cells. The AHV envelope glycoprotein, gp85, was found to be responsible for the cell-killing effect. Four independent lines of experimental evidence indicated that AHV induces a cytopathic effect through a typical programmed cell death, apoptosis: (i) morphological changes in cells visualized by light microscopy, (ii) nuclear condensation and fragmentation indicated by 4',6-diamidino-2-phenylindole staining, (iii) intranucleosomal degradation of DNA demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining, and (iv) flow cytometry analysis of the DNA content of the infected cells. Quiescent endothelial G0/G1 cells were much more sensitive to AHV-induced apoptosis than actively dividing cells, suggesting that the AHV ability to induce apoptosis is dependent on the proliferative state of the infected cells.

External ATP induces [3H]dopamine ([3H]DA) release in rat pheochromocytoma cells (PC-12 cells). The ATP-induced release is a saturable process with half-effective concentration of EC50 = 80 μM. ADP is a poor secretagogue of [3H]DA (one-sixth of ATP) and AMP is devoid of secretory capabilities. Adenosine and the non-hydrolyzable analogues of ATP, AppNHp and AppCp are ineffective as inducers of [3H]DA, release, or as inhibitors of the ATP-induced [3H]DA release. The most potent antagonist of ATP-induced release is Coomassie Blue (IC50 = 25 MM), compared to ADPβS (IC50 = 500 μM). The overall rank order of potency is ATP > ADP ≫ AMP > adenosine, which is characteristic of the P2-purinergic receptor. ATP-induced secretion is absolutely Ca2+ dependent, indicating an exocytotic process and is independent of Mg2+ (up to 2 mM) suggesting that the active species is not ATP4-. (a) The ATP-induced 45Ca2+ influx into the cells is in good correlation to ATP induction of release (IC50 = 80 and 90 μM, respectively) and is carried over to ADP which has a diminished ability to induce both release and 45Ca2+ influx. (b) Divalent cations (Ba2+ > Sr2+ > Ln3+ > Mn2+) replace Ca2+ and support ATP-induced release similar to their effectiveness in supporting bradykinin- and K+ (50 mM)-induced release in PC-12 cells (Weiss, C., Sela, D., and Atlas, D. (1990) Neurosci. Lett. 119, 241-245). Combined together the absolute requirement of [Ca2+]ex for release, inhibition of release by Gd3+ (IC50 = 100 μM), Ni2+, and Co2+ (IC50 = 1 mM), and support of release by Ba2+, Sr2+, and Mn2+, we suggest that ATP induces Ca2+ entry via ligand-operated Ca2+ channels as previously suggested for ATP in smooth muscle cells (Benham, C. D., Bolton, T. B., Byren, N. G., and Large, W. A. (1987) J. Physiol. (Lond.) 387, 473-488). No significant inhibition by 1 μM verapamil, 10 μM nifedipine, or 2 mM Cd2+ argues against ATP activation of voltage-dependent Ca2+ channels as similarly shown for ATP-induced [3H]noradrenaline release (Inoue, K., Nakazawa, K., Fujimoro, K., and Takanaka, A. (1989) Neurosci. Lett. 106, 294-299). Thus, the widely distributed ATP receptor might play an essential role in Ca2+ homeostasis of the cell by introducing Ca2+ into the cell via specific ligand-gated Ca2+ channels.

Bradykinin (BK), a nonapeptide acting at the B2-type BK-receptor, and depolarization with high KCl (50 mM), induce catecholamine secretion in pheochromocytoma cells (PC-12). The mechanism underlying the BK-induced release, which is absolutely Ca2+-dependent, is not yet understood. Alkaline metals, barium (Ba2+), strontium (Sr2+) and other metal cations, manganese (Mn2+) or lanthanum (La3+), support BK-induced [3H]noradrenaline ([3H]NA) release. The extent of supporting transmitter release is dependent upon the specificity of the extracellular cation, with rank order potency of: Ba2+ > Sr2+ > Ca2+ > Mn2+La3+. The same rank order potency was observed for supporting both BK- and K+-induced release. [3H]NA release in the presence of Ba2+ or Sr2+ was much greater than in the presence of Ca2+, and unlike with Ca2+ was not saturable at the highest concentration measured. La3+ and Mn2+ were significantly less effective than Ca2+ at supporting release. These results strongly suggest that extracellular Ca2+ entry is essential for release, and that BK mediates release via a receptor-operated Ca2+ channel.